Project Summary Antibodies have been tremendously successful cancer therapeutics partly because they can neutralize their antigen?s biological activity, but their inability to cross the plasma membrane has limited targets to secreted or membrane-associated antigens. One general approach for delivering proteins intracellularly has been to conjugate cargos to cell-penetrating peptides (CPPs), which are short poly-cationic peptides, to induce cellular uptake. However, CPPs suffer from two major limitations: poor cytoplasmic delivery due to endosome entrapment following uptake and a lack of any intrinsic tissue-specific targeting capability. Endosome entrapment can be addressed by using endosomolytic peptides (ELPs), which disrupt membranes in a pH- dependent manner, to induce endosome escape in conjunction with CPPs. Relatively few CPP-ELP pairs have been tested, though, and little is known about how the individual components interact and cooperate with each other. CPPs can be granted tissue-specificity by masking them with polyanionic sequences that dissociate solely in the presence of extracellular proteases specifically expressed in the tissue of interest. However, only one activatable CPP (aCPP) based on the polyarginine CPP has been designed thus far and only for delivery of fluorescent probes and nanoparticles. We have previously developed a small adaptor protein (pG) that can be site-specifically photo-crosslinked to the constant region of any off-the-shelf IgG while preserving the binding affinity of its variable region. By introducing CPP, ELP, or other peptide sequences into pG recombinantly, not only can additional functionalities can be tested in a high-throughput manner, but the cargo can be easily swapped out. The goal of this proposal is to leverage this technology to develop an aCPP-ELP pair that can delivery native IgGs into the cytoplasm of living cells to inhibit intracellular proteins. In Aim 1, I will create a library of pG variants containing different CPP-ELP pairs. The first peptide (CPP or ELP) will be introduced into pG recombinantly while the second will be conjugated to pG by using sortase A, a bacterial transpeptidase. In Aim 2, I will test the library for cytoplasmic delivery by using a self-assembling splitGFP reporter system in which the larger splitGFP half is cytoplasmically expressed while the smaller half is fused to pG. To demonstrate that delivered IgG-pG conjugates are functional, I will inhibit multidrug resistance- associated protein 1 (MRP1), an efflux pump associated with chemotherapy resistance, with QCRL3, a monoclonal IgG that robustly inhibits MRP1 activity once bound to one of its cytoplasmic domains. Finally, in Aim 3, I will test the well-characterized polyarginine aCPP as well as novel ones designed based on other CPPs used in the library for cell delivery dependent on matrix metalloproteinase-2/9 (MMP-2/9), which is highly expressed in tumors. Completion of this proposal will provide a new approach for inhibiting intracellular proteins in living cells and would form the basis for developing therapeutic intracellular antibodies. Optimal aCPP-ELP pairs could also be utilized to deliver large protein cargos for other applications.